Poor drug penetration is a major problem in cancer therapy. Fibrotic tumor stroma has been considered as one of the central barriers that hamper drug penetration into tumor tissue. Recent data indicate that our tumor penetrating peptide, iRGD, takes advantage of the stroma to penetrate tumor tissue. We have also found that iRGD inhibits metastasis formation, possibly by modifying the tumor microenvironment. This proposal aims to understand the mechanisms of stroma penetration and anti-metastatic activities of iRGD by focusing on carcinoma-associated fibroblasts (CAFs), the most abundant and critical component of tumor stroma. The iRGD peptide (CRGDK/RGPD/EC) contains an RGD tumor specific motif and a CendR motif (R/KXXR/K) that mediates tissue and cell penetration through binding to neuropilin-1 (NRP-1). iRGD triggers a NRP-1- and energy-dependent active bulk transport through tumors that allows compounds to penetrate efficiently into tumor tissue and cells. Therefore, attaching a drug to iRGD or simply co-injecting a drug with iRGD leads to vastly enhanced drug penetration and drug activities in tumor tissue with no enhanced toxicity to normal organs. Our recent data suggest a central role of CAFs in the iRGD tumor penetration activities. Time- dependent tumor homing studies have revealed that iRGD primarily penetrates tumor stroma and then gradually spreads into adjacent tumor cells. During this process, iRGD extensively accumulates into CAFs. In addition to utilizing CAFs as a mediator of tissue penetration, iRGD also appears to affect CAF functions and alter tumor microenvironment. Long-term treatment of tumor mice with iRGD resulted in a dramatic decrease in the number of CAFs in the tumor tissue and a significant inhibition of metastasis. Interestingly, it is becoming evident that the expression of NRP-1, the receptor of iRGD, is critical for activated fibroblast functions. In this application, we propose to study the roles of NRP-1 positive CAFs in the tumor penetration mechanism and the biological activities of iRGD. Mechanistic studies of the iRGD tumor stroma penetration would facilitate the use of the technology in targeting desmoplastic tumors such as pancreatic cancer that are the most therapeutically challenging tumor types in the clinic. Studying iRGD effects on tumor microenvironment modification may lead to the discovery of novel biological functions of iRGD that may serve as a basis of metastasis treatment strategies. Major advances in cancer therapy may ensue.